Real-time　in　situ　monitoring　of　low-abundance　cancer　biomarkers　(e.g.,　miRNAs　and　proteins)　in　living　cells　by　nonenzymatic　assembly　entirely　from　original　DNA　probes　remains　unexplored　due　to　an　extremely　complex　intracellular　environment.　Herein,　a　nonenzymatic　palindrome-catalyzed　DNA　assembly　(NEPA)　technique　is　developed　to　execute　the　in　situ　imaging　of　intracellular　miRNAs　by　assembling　a　three-dimensional　nanoscale　DNA　spherical　structure　(NS)　with　low　mobility　from　three　free　hairpin-type　DNAs　rather　than　from　DNA　intermediates　based　on　the　interaction　of　designed　terminal　palindromes.　Target　miRNA　was　detected　down　to　1.4　pM,　and　its　family　members　were　distinguished　with　almost　100%　accuracy.　The　subcellular　localization　of　NS　products　can　be　visualized　in　real　time.　The　NEPA-based　sensing　strategy　is　also　suitable　for　the　intracellular　in　situ　fluorescence　imaging　of　cancer-related　protein　receptors,　offering　valuable　insight　into　developing　sensing　protocols　for　understanding　the　biological　function　of　vital　biomolecules　in　disease　pathogenesis　and　future　therapeutic　applications.　Copyright　©　2020　American　Chemical　Society.